Sequencing by orthogonal synthesis

Abstract

A method for sequencing includes steps of (a) providing first and second nucleic acid templates, wherein the two templates have different sequences; (b) extending a first primer bound to the first template using a first polymerase species and a first set of nucleotide analogs; (c) extending a second primer bound to the second template using a second polymerase species and a second set of nucleotide analogs, wherein the first polymerase species is different from the second polymerase species and wherein the first set of nucleotide analog is different from the second set of nucleotide analog, (d) detecting the first and second primer extension products; and (e) repeating steps (b) through (d), thereby determining the different sequences of the first and second templates.

What is claimed is:  
     
       1. A method for sequencing nucleic acid templates, comprising
 (a) providing an array of sites, wherein each site comprises a first nucleic acid template and a second nucleic acid template, 
 wherein the first nucleic acid template has a sequence that is different from the sequence of the second nucleic acid template; 
 (b) extending a DNA primer bound to the first template using a DNA polymerase species and a set of deoxyribonucleotide analogs, thereby producing a DNA primer extension product comprising a deoxyribonucleotide analog at each of the sites; 
 (c) extending an RNA primer bound to the second template using an RNA polymerase species and a set of ribonucleotide analogs, thereby producing an RNA a primer extension product comprising a ribonucleotide analog at each of the sites; 
 (d) detecting the DNA primer extension product and the RNA primer extension product at each of the sites; and 
 (e) repeating steps (b) through (d), thereby determining the different sequences of the first template and the second template at each of the sites. 
 
     
     
       2. The method of  claim 1 , wherein the detecting uses a detector having a spatial resolution that is too low to resolve points at distance equivalent to the spacing between the DNA primer extension product and the RNA primer extension product at each of the sites. 
     
     
       3. The method of  claim 2 , wherein the detector is an optical detector. 
     
     
       4. The method of  claim 3 , wherein the nucleotide analogs comprise optical labels. 
     
     
       5. The method of  claim 4 , wherein the optical labels of the set of deoxyribonucleotide analogs are different from the optical labels of the set of ribonucleotide analogs. 
     
     
       6. The method of  claim 3 , wherein a subset of the nucleotide analogs in the set of deoxyribonucleotide analogs comprise optical labels. 
     
     
       7. The method of  claim 6 , wherein a subset of the nucleotide analogs in the set of ribonucleotide analogs comprise optical labels. 
     
     
       8. The method of  claim 7 , wherein the set of deoxyribonucleotide analogs comprise optical labels that are different from the optical labels of the set of ribonucleotide analogs. 
     
     
       9. The method of  claim 8 , wherein the set of deoxyribonucleotide analogs comprise only one type of optical label that is detected in step (d) and the set of ribonucleotide analogs comprise only one type of optical label that is detected in step (d). 
     
     
       10. The method of  claim 9 , wherein the one type of optical label is attached to substantially all of the nucleotide analogs of a first species in the set of deoxyribonucleotide analogs,
 the one type of optical label is attached to a subset of the nucleotide analogs of a second species in the set of deoxyribonucleotide analogs, 
 substantially all of the nucleotide analogs of a third species in the set of deoxyribonucleotide analogs are attached to a ligand, and 
 substantially all of the nucleotide analogs of a fourth species in the set of deoxyribonucleotide analogs are not attached to the one type of optical label or to the ligand. 
 
     
     
       11. The method of  claim 8 , wherein the set of deoxyribonucleotide analogs comprise only two types of optical labels that are detected in step (d) and the set of ribonucleotide analogs comprise only two types of optical labels that are detected in step (d). 
     
     
       12. The method of  claim 11 , wherein only one of the two types of optical labels is attached to substantially all of the nucleotide analogs of a first species in the set of deoxyribonucleotide analogs,
 only a second of the two types of optical labels is attached to substantially all of the nucleotide analogs of a second species in the set of deoxyribonucleotide analogs, 
 the one of the two types of optical labels and the second of the two types of optical labels are attached to nucleotide analogs of a third species in the set of deoxyribonucleotide analogs, and 
 substantially all of the nucleotide analogs of a fourth species in the set of deoxyribonucleotide analogs are not attached to the one of the two types of optical labels or the second of the two types of optical labels. 
 
     
     
       13. The method of  claim 2 , wherein a pixel of the detector acquires signals from both the DNA primer extension product and the RNA primer extension product. 
     
     
       14. The method of  claim 1 , wherein the first nucleic acid template comprises at least one base moiety that is the same species as a base moiety in the second nucleic acid template. 
     
     
       15. The method of  claim 14 , wherein the first nucleic acid template and the second nucleic acid template comprise DNA. 
     
     
       16. The method of  claim 15 , wherein the at least one base moiety is selected from the group consisting of adenine, thymine, cytosine, and guanine. 
     
     
       17. The method of  claim 1 , wherein steps (b) and (c) are carried out simultaneously. 
     
     
       18. The method of  claim 1 , wherein steps (b) and (c) are carried out sequentially. 
     
     
       19. The method of  claim 1 , wherein a single nucleic acid molecule contains the first nucleic acid template and the second nucleic acid template. 
     
     
       20. The method of  claim 1 , wherein the first nucleic acid template and the second nucleic acid template are on different nucleic acid molecules. 
     
     
       21. The method of  claim 1 , wherein the sites have an area that is no greater than 100 μm 2 . 
     
     
       22. The method of  claim 1 , wherein the sites comprise multiple copies of the first nucleic acid template and the second nucleic acid template. 
     
     
       23. The method of  claim 22 , wherein the multiple copies comprise a nucleic acid cluster. 
     
     
       24. The method of  claim 1 , wherein the DNA polymerase is selective for the first template compared to the second template and wherein the RNA polymerase is selective for the second template compared to the first template. 
     
     
       25. A method for sequencing nucleic acid templates, comprising
 (a) providing a first nucleic acid template and a second nucleic acid template, 
 wherein the first nucleic acid template has a sequence that is different from the sequence of the second nucleic acid template; 
 (b) extending a DNA primer bound to the first template using a DNA polymerase species and a set of deoxyribonucleotide analogs, thereby producing a DNA primer extension product comprising a deoxyribonucleotide analog; 
 (c) extending an RNA primer bound to the second template using an RNA polymerase species and a set of ribonucleotide analogs, thereby producing an RNA primer extension product comprising a ribonucleotide analog, 
 (d) detecting the DNA primer extension product and the RNA primer extension product using a detector having a resolution that is lower than the spatial separation between the DNA primer extension product and the RNA primer extension product; and 
 (e) repeating steps (b) through (d), thereby determining the different sequences of the first template and the second template. 
 
     
     
       26. The method of  claim 25 , wherein the detector is an optical detector, and wherein the nucleotide analogs comprise optical labels. 
     
     
       27. The method of  claim 26 , wherein the optical labels of the set of deoxyribonucleotide analogs are different from the optical labels of the set of ribonucleotide analogs. 
     
     
       28. The method of  claim 25 , wherein a subset of the nucleotide analogs in the set of deoxyribonucleotide analogs comprise optical labels. 
     
     
       29. The method of  claim 28 , wherein a subset of the nucleotide analogs in the set of ribonucleotide analogs comprise optical labels. 
     
     
       30. The method of  claim 29 , wherein the set of deoxyribonucleotide analogs comprise optical labels that are different from the optical labels of the set of ribonucleotide analogs. 
     
     
       31. The method of  claim 25 , wherein the first nucleic acid template comprises at least one base moiety that is the same species as a base moiety in the second nucleic acid template. 
     
     
       32. The method of  claim 25 , wherein steps (b) and (c) are carried out simultaneously. 
     
     
       33. The method of  claim 25 , wherein steps (b) and (c) are carried out sequentially. 
     
     
       34. The method of  claim 25 , wherein a single nucleic acid molecule contains the first nucleic acid template and the second nucleic acid template. 
     
     
       35. The method of  claim 25 , wherein the first nucleic acid template and the second nucleic acid template are on different nucleic acid molecules. 
     
     
       36. The method of  claim 25 , wherein the sites comprise multiple copies of the first nucleic acid template and the second nucleic acid template.